Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy

Abstract

Leber congenital amaurosis (LCA) is a rare hereditary retinal degeneration caused by mutations in more than a dozen genes. RPE65, one of these mutated genes, is highly expressed in the retinal pigment epithelium where it encodes the retinoid isomerase enzyme essential for the production of chromophore which forms the visual pigment in rod and cone photoreceptors of the retina. Congenital loss of chromophore production due to RPE65-deficiency together with progressive photoreceptor degeneration cause severe and progressive loss of vision. RPE65-associated LCA recently gained recognition outside of specialty ophthalmic circles due to early success achieved by three clinical trials of gene therapy using recombinant adeno-associated virus (AAV) vectors. The trials were built on multitude of basic, pre-clinical and clinical research defining the pathophysiology of the disease in human subjects and animal models, and demonstrating the proof-of-concept of gene (augmentation) therapy. Substantial gains in visual function of clinical trial participants provided evidence for physiologically relevant biological activity resulting from a newly introduced gene. This article reviews the current knowledge on retinal degeneration and visual dysfunction in animal models and human patients with RPE65 disease, and examines the consequences of gene therapy in terms of improvement of vision reported.

Figure 1

Photoreceptor layer thickness topography in patients with RPE65-LCA. (A, B) Cross-sectional scans along the vertical meridian in a 15-year-old normal subject and a 10-year-old patient (P4) with RPE65-LCA. Hyporeflective layer corresponding to the outer nuclear layer (ONL) is shown. (C) Normal topography of ONL thickness as an average map based on a group of six normal subjects. (D, E) Patients at 6 and 10 years of age compared with (F, G) two patients in their early 20s. Traces of major blood vessels and location of the optic nerve head are overlaid on each map (depicted as right eyes). T, temporal; N, nasal; S, superior; I, inferior. Bottom left: color scale for ONL thickness. Reprinted from Jacobson et al., 2008, copyright © held by the Association for Research in Vision and Ophthalmology.

Figure 2

Foveal cone morphology of young RPE65-LCA patients. (A) Cross-sectional scans along the horizontal meridian of the central retina of a normal child (upper panel) and three children with LCA due to RPE65 mutations (lower panels). Arrows and brackets indicate ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segment; OS, outer segment. (B) Mean foveal ONL, IS and OS thickness in a group of young subjects with normal vision (ages 5–15 years) and in eight young patients with RPE65–LCA (ages 6–20 years). Mean values for the parameters from the RPE65–LCA group are also shown (Avg). Error bars represent +/−2 SD. Reprinted from Maeda et al., 2009, by permission from Oxford University Press.

Figure 3

Retinal function in the Rpe65^−/− mouse model of RPE65-LCA. (A) Dark-adapted ERGs evoked by increasing intensities of blue light stimuli (shown to the left of the traces) in a representative Rpe65^−/− mouse show an elevated b-wave threshold compared to Rpe65^+/+. (B) A physiologically based model (smooth lines) is fit to the leading edges (initial 4–15 ms depending on response) of dark-adapted ERG photoresponses (symbols) evoked by 3.6 and 2.2 log scot-cd.s.m⁻² flashes to quantify the activation phase of rod phototransduction. Rpe65^−/− mouse photoresponses show smaller saturated amplitude and a slower initial slope. (C) Photoresponses in three Rpe65^+/+ mice are compared to a group of Rpe65^−/− mice. Lines are the model of rod phototransduction activation fitted to a pair of photoresponses; only maximal responses are shown for clarity. (D) Maximum amplitude and sensitivity parameters of ERG photoresponses in dark-adapted Rpe65^−/− mice are significantly (**) different than the results in Rpe65^+/+ mice. Error bars (1 SEM) are smaller than symbols for some data. Modified from van Hooser et al., 2000, copyright © by the National Academy of Sciences.

Figure 4

Retinal function in the Rpe65^rd12 mouse model of RPE65-LCA. (A) Dark adapted ERGs evoked by increasing stimulus intensities (shown to the left of key traces) for representative 2-month-old wt and Rpe65^rd12 mice. Blue flashes were used for all intensities except the highest, which were evoked by white flashes. Traces start at stimulus onset. (B) A physiologically based model of phototransduction (smooth lines) is fit as an ensemble to the leading edges of ERG photoresponses (symbols) evoked by 3.6 and 2.2 log scot-cd.s.m⁻² flashes. The response from the mutant shows reduced amplitude and a slower initial slope. (C) Summary statistics of maximum amplitude (R_max) and sensitivity (log S) parameters obtained from photoresponse modeling in Rpe65^rd12 mice are significantly (**) different than wt. (D) Luminance-response functions derived from ERG b-wave series show diminished light sensitivity in mutant animals indicated by a shift to the right of the curves. Mutant animals also show a reduction in maximum amplitude. Error bars are 1SD. Reprinted from Roman et al., 2007b, copyright © by Molecular Vision.

Figure 5

Electroretinographic (ERG) abnormalities in a representative RPE65-mutant dog. (A) ERGs evoked by standard white flashes (0.4 log scot-cd.s.m⁻²) presented under dark-adapted (DA, black traces) and light-adapted (LA, red traces) conditions. DA traces are single flashes, LA traces are averages obtained at repetition frequencies of 1 (top) and 29 Hz (bottom). Black vertical lines show the timing of the flashes. (B) ERG photoresponses evoked by white flashes of high energy (3.7 log scot-cd.s.m⁻²) under DA and LA conditions, same data are shown on slow (top) and fast (bottom) time scales to allow interpretation of late and early components, respectively. Gray lines show the baseline and the 4-ms time point at which rod and cone photoreceptor responses were measured. Modified from Acland et al., 2005.

Figure 6

Standard ERGs in patients with RPE65-LCA. Rod, mixed cone–rod, and cone ERGs from six representative patients with RPE65–LCA (age range, 5-23 years) were compared with ERGs of a patient with a rhodopsin (R135L) gene mutation showing residual, severely abnormal, cone-mediated function. A normal subject (age 20 years) is shown for comparison (left). Traces start at stimulus onset except for 29 Hz where the timing of stimuli is shown with vertical gray lines; calibrations are to the right and below waveforms. ERGs can be undetectable, even at early ages. Detectable ERGs show a pattern similar to that of the patient with a RHO mutation and severe rod dysfunction but residual and abnormal cone function. Modified from Jacobson et al., 2009, copyright © held by the Association for Research in Vision and Ophthalmology.

Figure 7

Cone-photoreceptor mediated visual function in RPE65-LCA. (Upper) Dark-adapted psychophysical sensitivities to chromatic and achromatic stimuli presented across the horizontal meridian in two young adult RPE65-LCA patients (P14, P15). Sensitivities to chromatic stimuli (500 nm blue, 650 nm red) are shown on a common axis of radiometric equivalence, and sensitivities to achromatic (white) stimuli are vertically shifted to match the 500 nm stimulus results. Mean normal dark adapted rod sensitivity to the 500 nm stimulus and dark-adapted cone sensitivity to 650-nm stimulus obtained during the cone plateau are shown (gray lines labeled Rod and Cone). (Lower) Comparison of the difference in chromatic sensitivity at each locus (symbols) to predicted difference for rod or cone mediation (dashed lines) based on spectral sensitivities of normal rod- and cone-mediated vision. Physiological blind spot is shown as a hatched bar. Temp, temporal; Nas, nasal. Modified from Jacobson et al., 2007a, copyright © by the National Academy of Sciences.

Figure 8

Mean cortical signal change in response to visual stimulation in human RPE65-LCA (n=6) and normal control populations (n=8). (A and B) The BOLD fMRI response is shown for each population at two stimulus intensities: (A) −3 log and (B) at/near maximum (between −1.2 log and 0 log). The areas of response are displayed upon a digitally inflated right hemisphere. Sulci are indicated in dark gray and gyri in light gray. (Insets) The general position of several retinotopic and higherorder visual areas, derived from data from control participants, is shown. (C) Cortical activation as a function of stimulus luminance is presented. The volume of posterior cortical tissue demonstrating a substantial (>2%) response shows a sigmoidal relationship to the strength of visual stimulation in normal controls and in patients. A Hill function (gray smooth lines) is fit by eye to the data points corresponding to each participant. Reprinted from Aguirre et al., 2007.

Figure 9

Retinas of patients with RPE65-LCA can have more photoreceptor nuclear layer than predicted from vision. (A) Foveal outer nuclear layer (ONL) thickness as a function of dark-adapted cone-mediated sensitivity (650 nm). (B and C) ONL thickness as a function of dark-adapted sensitivity (500 nm) at 3.6 mm in temporal (B) and superior (C) retina. Rod, rod-mediated sensitivity; Cone, cone-mediated sensitivity; Pts, patients without RPE65 mutations. Normal variability is described by the ellipses encircling the 95% confidence interval of a bivariate Gaussian distribution. Dotted lines define the idealized model of the relationship between retinal structure and function in pure photoreceptor degenerations and the region of uncertainty that results by translating the normal variability along the idealized model. (Inset) Retinal location (white arrow on fundus image) of colocalized measures of structure and function. Overlaid onto the fundus image are cone density (a) and rod density (b and c) along horizontal and vertical meridia. Modified from Jacobson et al., 2005, copyright © by the National Academy of Sciences.

Figure 10

Improvement of retinal function by gene therapy in Rpe65^−/− mice performed either in utero or at the post natal age of 1-2.5 months. (a) Dark-adapted electroretinograms (ERGs) from the two eyes (Eye 1, Eye 2) of a representative Rpe65^−/− mouse 2 months after a monocular in utero subretinal injection of AAV2/1-CMV-hRPE65. ERGs were evoked by increasing intensities of blue light stimuli (stimulus luminance is to the left of key traces). Traces start at stimulus onset. ERG waveforms from Eye 1 are severely abnormal (elevated bwave threshold of 3-4 log units) and resemble those from uninjected Rpe65^−/− mice. Eye 2 responses are dramatically different and more like those of an age matched normal mouse (left column, for comparison). The b-wave threshold is near normal and there are sizeable but abnormal amplitudes; an a-wave can be detected at the brighter intensities. (b) A physiologically-based model of rod phototransduction activation is fit as an ensemble to the leading edges (first 4-12 ms after stimulus) of dark-adapted photoresponses (symbols) evoked in the eyes of the Rpe65^−/− mouse shown in (a); normal photoresponses to the same stimuli are shown on the left. (c and d) Retinal function results from all animals in all groups. For in utero-injected animals (left), Eye 1 is defined as the eye with the lower photoresponse sensitivity. Red-filled symbols (“treatment success”) represent those results falling beyond the 99% confidence interval limit (upper boundary of the gray bars) for each parameter determined from uninjected age-matched Rpe65^−/− mice. Lines connect data obtained from the two eyes of each animal. Many of the Eye 2 group show retinal function that is as good as or better than that in eyes treated postnatally (Tx, right). Reprinted from Dejneka et al., 2004.

Figure 11

Electroretinography parameters of Rpe65^rd12 mice to different doses of subretinal AAV2-hRPE65. (A) ERGs evoked by 0.1 log scot-cd.s.m⁻² flashes (upper row) and by 3.6 log scot-cd.m.s⁻² flashes (lower row) in treated (colors) and untreated (gray) eyes of one Rpe65^rd12 mouse from each dose group (0.01X-1X). As vector dose increases, responses become asymmetric with treated retinas showing increasing amplitude of b-waves and faster photoresponses. Photoreceptor activation models (smooth lines) fit to the photoresponses are shown. All traces start at stimulus onset. (B) Photoresponse parameters in Rpe65^rd12 eyes treated with a range of vector doses. As dosage increases above 0.01X, parameter pairs drift outside of the 99% confidence region (dashed ellipse) defined by the untreated eyes of Rpe65^rd12 animals and start approaching wt levels. (C) Luminance response parameters in treated Rpe65^rd12 eyes similarly show a dose-related progression from the region corresponding to untreated eyes to the region corresponding to wt eyes. Reprinted from Roman et al., 2007, copyright © by Molecular Vision.

Figure 12

Long term restoration of vision in RPE65-mutant dogs by gene therapy. (A) ERGs evoked by standard white flashes in the right eye of an RPE65-mutant dog before treatment (Pre-Tx) and over a 3-year interval after treatment. Flashes presented under dark-adapted (DA) and light-adapted (LA) conditions. DA traces are single waveforms, LA traces are averages obtained at repetition frequencies of 1 (top) and 29 Hz (bottom). Black vertical lines show the timing of the flashes. (B) ERG photoresponses evoked with white flashes of high energy over the same 3-year interval in the same eye as in A. Waveforms displayed as in A. (C) Two eyes with subretinal AAV-RPE65 (green symbols) show stable level of partial restoration of retinal rod and cone function, whereas two eyes with intravitreal AAV-RPE65 (gray symbols) show amplitudes similar to those of untreated eyes. Horizontal dashed lines represent the upper limit (mean + 3 SD) of the respective measurement in the group of control RPE65-mutant affected eyes, which had not received treatment. Modified from Acland et al., 2005.

Figure 13

Brainstem responses in the dog using the pupillary light reflex show gene therapy treatment effect. (Left panel) A representative RPE65-mutant dog pre- and 1 month post-treatment (video frames show the pupil before and 0.6 s after a 0.6 log scot-cd.m⁻² stimulus; pupillary margin delineated). (Middle panel) Pupillary contraction amplitude and timing in this eye post-treatment (unfilled triangles) was within normal limits (gray band); there was no response pre-treatment (filled triangles). (Right panel) Threshold and amplitude parameters show treatment success in RPE65-mutant eyes after gene therapy compared to untreated/pretreatment results. Modified from Aguirre et al., 2007.

Figure 14

Visual function improvement in RPE65-LCA patients after gene therapy is stable up to 12 months after treatment. (A) Light sensitivity to achromatic stimuli in study eyes along vertical (P1 and P2) and horizontal (P3) meridians after allowing for an extended (3–8 hr) period of dark adaptation. Sensitivity values recorded post-treatment (colored symbols), are compared with the mean baseline values recorded before treatment (gray symbols). Images of the ocular fundus of the study eyes obtained at 12 months after treatment. All images are shown as left retina for clarity and comparability. F, fovea. (B) Light sensitivity measures with chromatic stimuli support stability of rod and cone photoreceptor-based vision 12 months after treatment in retinal regions of peak response to gene therapy. Rod function measured with blue stimuli after standard (Std) or extended dark adaptation (Ext-DA) conditions. Cone function measured with red stimuli after Ext-DA conditions (at the fovea) or during the cone plateau period in the dark after light adaptation (at extrafoveal locations). The sensitivity axes are shifted vertically to match red and blue stimuli for cone-mediated detection in patients. I, inferior; S, superior; T, temporal retina. Reprinted from Cideciyan et al. 2009a, copyright © by Mary Ann Liebert, Inc..

Figure 15

Fixation location and fixation stability of control and study eyes in RPE65-LCA patients do not change after gene therapy treatment. (A) Pairs of traces show the x- and y-axis deflections of the location of the fovea recorded with infrared retinal imaging over 10-s epochs in the study eyes of three patients at baseline and days 30, 60, and 90 after treatment. Control eyes showed similar results (not shown). Horizontal dashed lines are the reference location of the fovea at the start of the recording session 15–35 s preceding the selected epoch. All patients show abnormal eye movements with horizontal and vertical components of instability and larger amplitude jerk nystagmus occurring at frequencies of ~1–2 Hz. All plots are shown as equivalent left eyes for clarity. (B) Traces from a representative normal subject. (C) Summary of fixation instability in all subjects. Horizontal dashed line is a conservative (+3 SD) upper limit of normal (N) from the mean value. Study and control eyes of patients 1, 2, and 3 (P1, P2, P3) at two baseline (B) visits show fixation instability similar to other patients with RPE65-LCA (dark hexagons). At posttreatment days 30, 60, and 90, there are no consistent increases or decreases in fixation instability for the study and control eyes. Reprinted from Cideciyan et al. 2008, copyright © by the National Academy of Sciences.

Figure 16

Dark adaptation kinetics across the retinal region of study eyes in RPE65-LCA patients (P2 and P3) after gene therapy. Rod- and cone-photoreceptor-mediated visual function is measured with chromatic stimuli after a 7 log scot-td.s yellow adapting flash (presented at time 0) in patient 2 (at 3.6 and 7.2 mm inferior loci) and patient 3 (at 17 mm temporal locus). Also shown are detailed results from one normal (N) subject at 3.6 mm inferior (Left) and mean results from normal subjects at each location (gray lines). Cone adaptation kinetics (red symbols) are fast and do not show a difference from healthy cones. Rod adaptation kinetics (blue symbols) are extremely slow compared with healthy rods, and in patient 2 there is evidence for a large intraretinal difference in recovery rate. Additionally, absolute thresholds (arrow along the ordinate) of both rod and cone systems are abnormally elevated. Modified from Cideciyan et al. 2008, copyright © by the National Academy of Sciences.

Figure 17

Pupillary responsivity of RPE65-LCA patients increases after gene therapy. (A) Change in pupil diameter evoked by a short duration light stimulus (0.1 s, −0.6 log scot-cd.m⁻², green, dark-adapted) in study and control eyes of the three RPE65-LCA patients at 18 months before treatment (thin black traces) and 1 month after treatment (red, study eye; blue, control eye) compared with results from normal subjects (gray band, mean+/−2 SEM; n=13). Pretreatment eyes do not show changes in pupil diameter that are distinguishable in magnitude (<0.3 mm) from spontaneous oscillations of the pupil diameter of the dark-adapted eye. Posttreatment (red) traces show sizable pupil contractions in the study eyes of two of the patients (patients 2 and 3) but not in patient 1. Pupils from control eyes (blue traces) after treatment do not contract in response to this stimulus. Stimulus monitor trace at the bottom left. (Inset) Video frames of pupils of control and study eyes of patient 2 at 0.9 s after the light stimulus; pupillary margin is outlined in white (pretreatment), red (post-treatment, study eye), or blue (posttreatment, control eye) for visibility; calibration bar at the bottom right. (B) Luminance response functions measured at 0.9 s after stimulus onset in both eyes of each patient before treatment (thin black lines) and after treatment in the study eye (red) compared with the control eye (blue) and with normal data (mean+/−SEM, white symbols). Before treatment (thin black lines), pupil contractions were observed only with the two highest luminance stimuli. After treatment (red symbols), there were contractions in the study eye of patients 2 and 3 within a range of stimuli and there was a shift of the luminance response function to the left; no such responding was recorded in patient 1 or in the control eyes. Arrow points to stimulus luminance of the waveforms shown in A. (C) Pupillary response thresholds in patients with untreated RPE65-LCA (gray hexagons, other RPE65-LCA patients; gray squares, patient 1; gray circles, patient 2; gray diamonds, patient 3) show abnormalities in excess of 5 log units compared with normal (white hexagon, mean+/−2 SD). Thresholds in the study subjects before treatment are similar to each other and to other patients with RPE65-LCA (gray hexagons, n=3; gray line, mean+/−2 SD for untreated RPE65-LCA). The treated eye of patient 2 improved by 1.6 log units, from 0.74 log scot-cd.m⁻² before treatment to −0.86 log scot-cd.m⁻² after treatment (red). Patient 3 improved by 1.8 log units from 0.83 log scot-cd.m⁻² before treatment to −0.92 log scot-cd.m⁻² after treatment. The untreated eyes did not change (blue symbols). Patient 1 showed no detectable changes, which may be attributed to the smaller increase in posttreatment visual sensitivity compared with patients 2 and 3. Reprinted from Cideciyan et al. 2008, copyright © by the National Academy of Sciences.

Figure 18

Slow emergence of a pseudo-fovea in an RPE65-LCA patient within the treated retinal region and perception of previously unseen stimuli. Panel (a) shows the eye (upper image) and the patient’s retina (lower image). Overlaid contours of constant sensitivity (measured by means of microperimetry) show no change in visual sensitivity between 1 and 12 months after treatment. F denotes the fovea, and ST the superotemporal retina that received treatment. The circular pattern is a standard grid centered on the fovea. Retinal distance calibration corresponding to 5 degrees of visual angle is shown. Panel (b) shows fixation clouds (scatter plots) in the study eye of the patient at baseline and the statistics of fixation dwell time (bar graphs) along the diagonal meridian as a function of the target luminance. All three luminances were perceived by the patient at all visits. At the 2.7- and 2.4-log₁₀ luminances, fixation was within 3 degrees of the fovea more than 99% of the time at all visits except at the 12-month visit for 2.4-log₁₀ luminance, when 68% of fixation time dwelled in an ST retinal region 4 to 9 degrees from the fovea. At 2.1-log₁₀ luminance, fixations showed increasingly greater excursions into the ST retina between 2 and 9 months after treatment. At 12 months, 89% of fixation time dwelled in the ST region 4 to 9 degrees from the fovea. Thin vertical lines represent the foveal location. Red bars indicate significant (>3 degrees) excursions from the fovea. Panel (c) shows that a dimmer target (1.8 log₁₀) was not perceived by the patient’s study eye during baseline though 9 months after treatment. At 12 months, this stimulus was perceived for the first time with a coincident shift of fixation into the ST retinal region. Reprinted from Cideciyan et al., 2009b. Copyright © 2009 Massachusetts Medical Society. All rights reserved.